Synthesis and cytotoxic activity of biphenylurea derivatives containing indolin-2-one moieties

Article abstract of DOI:10.3390/molecules21060762

In our endeavor towards the development of potent anticancer agents, two different sets of biphenylurea-indolinone conjugates, 5a-s and 8a,b were synthesized. The in vitro cytotoxicity of the synthesized compounds was examined in two human cancer cell lin

Full text of DOI:10.3390/molecules21060762

molecules
Article
Synthesis and Cytotoxic Activity of Biphenylurea
Derivatives Containing Indolin-2-one Moieties
Wagdy M. Eldehna 1,*, Mohamed Fares 1,2,†, Hany S. Ibrahim 1,†, Muhammad A. Alsherbiny 3,†
Mohamed H. Aly 4,5, Hazem A. Ghabbour 6 and Hatem A. Abdel-Aziz 7
,
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City,
Cairo 11829, Egypt; ph.fares@yahoo.com (M.F.); hany.s.ibrahim@gmail.com (H.S.I.)
School of Chemistry, University of Wollongong, Wollongong 2522, New South Wales, Australia
Department of Pharmacognosy, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, Cairo 11562,
2
3
Egypt; Muhammad.alsherbiny@pharma.cu.edu.eg
Department of Pharmacology and Toxicology, Faculty of Pharmacy, British University in Egypt, Cairo 11562,
Egypt; mohamed.aly@bue.edu.eg
Department of Biology, The American University in Cairo, New Cairo 11835, Egypt
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457,
Riyadh 11451, Saudi Arabia; ghabbourh@yahoo.com
Department of Applied Organic Chemistry, National Research Center, Dokki, Giza 12622, Egypt;
hatem_741@yahoo.com
4
5
6
7
*
†
Correspondence: wagdy2000@gmail.com; Tel.: +20-2-2860-9114; Fax: +20-2-2860-9117
These authors contributed equally to this work.
Academic Editors: Jean Jacques Vanden Eynde and Annie Mayence
Received: 12 April 2016; Accepted: 3 June 2016; Published: 10 June 2016
Abstract: In our endeavor towards the development of potent anticancer agents, two different sets of
biphenylurea-indolinone conjugates, 5a–s and 8a,b were synthesized. The in vitro cytotoxicity of the
synthesized compounds was examined in two human cancer cell lines, namely MCF-7 breast cancer
and PC-3 prostate cancer cells using the sulforhodamine B (SRB) colorimetric assay. In particular,
the MCF-7 cancer cell line was more susceptible to the synthesized compounds. Compound 5o
(IC50 = 1.04 ˘ 0.10 µM) emerged as the most active member in this study against MCF-7, with 7-fold
increased activity compared to the reference drug, doxorubicin (IC50 = 7.30
˘
0.84
µ
M). Compounds
0.17,
M, respectively. All of the tested compounds were filtered according
to the Lipinski and Veber rules and all of them passed the filters. Additionally, several ADME
descriptors for the synthesized compounds 5a and 8a were predicted via a theoretical kinetic
study performed using the Discovery Studio 2.5 software.
5l
3.87
,
5q and 8b also exhibited superior cytotoxic activity against MCF-7 with IC50 values of 1.93
0.31 and 4.66 0.42
˘
˘
˘
µ
–
s
,b
Keywords: biphenylurea; cytotoxic activity; indolinone; ADME
1. Introduction
According to the World Health Organization (WHO) estimates, cancer is a disease responsible
for major morbidity and mortality worldwide. As reported in 2012 it resulted in about 8.2 million
deaths [1]. Owning to its high incidence, prevalence, morbidity as well as mortality rates, cancer has
been identified as a huge burden on the society. According to the statistics provided by GLOBOCAN in
2012, around 14.1 million newly diagnosed cancer cases were recorded. Among the newly diagnosed
cases in females, breast cancer ranked in the first position worldwide and was regarded as the main
cause of deaths attributed to cancer. On the other hand, prostate cancer was reported to be of the
highest incidence among males in developed countries [2]. Although extensive research work had
been conducted to synthesize and evaluate novel anticancer agents [
3
–8
], satisfactory progress, relative
Molecules 2016, 21, 762; doi:10.3390/molecules21060762
www.mdpi.com/journal/molecules

Molecules 2016, 21, 762
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to the speed of the disease progression, towards the development of anticancer agents of optimum
activity and minimum side effects is not yet been accomplished.
Biphenylurea derivatives showed interesting activity as anticancer agents. For example, sorafenib,
(
1
, Figure 1) is a potent diphenylurea VEGFR-2 inhibitor in addition to its inhibitory action on c-Kit,
PDGFR , and Raf kinases (Figure 1) [ ]. The U.S. FDA has approved it for the treatment of advanced
renal cell carcinoma (RCC) and for hepatocellular carcinoma (HCC) [10]. Regorafenib ( , Figure 1), a
fluoro derivative of sorafenib, [11] inhibits angiogenic kinases VEGFR-1/3, PDGFR , FGFR1, and Tie-2.
β
9
2
β
Furthermore, it showed anti-proliferative activities on different cancer cell lines [12]. Regorafenib was
approved for previously treated metastatic colorectal cancer (mCRC) and advanced gastrointestinal
stromal tumors (GIST) [13]. Linifanib, (
trials, has selective inhibitory action on VEGFR and PDGFRs in patients with locally advanced or
metastatic non-small cell lung cancer [14]. SLC-0111 ( ) is a low molecular weight biphenylurea
3, Figure 1), a novel biphenylurea derivative in phase II clinical
4
derivative in phase I clinical trials which has shown significant antitumor/ antimetastatic activity in
animal models of the disease via the inhibition of tumor-associated human carbonic anhydrase (hCA)
IX and XII isoforms (Ki = 4.5 and 45 nM, respectively) [15]. Its thioureido analog
5 displayed selective
inhibition against tumor-associated hCA IX and hCA XII isoforms with Ki = 39, 4 nM, respectively [16].
Figure 1. Biphenylurea derivatives and indolinone derivatives as anticancer agents and the proposed
structures 5a–s and 8a,b.
On the other hand, the indolinone moiety proved its success in many compounds as
antineoplastic [17,18]. This success is supported by the FDA approval of the use of indolinone
derivative, sunitinib maleate (20a) (Sutent®) in combating advanced renal carcinoma [19] and
gastrointestinal stromal tumors [20]. In addition, toceranib phosphate (20b) [21] (Palladia®), an
orally bioavailable compound, is approved by FDA for the treatment of cutaneous (skin-based) mast
cell tumors. On the other hand there are many compounds derived from indolinone are in clinical
trials, for example, SU5416 (Semaxanib, 21a) is an indolinone derivative in clinical trials, it showed
extreme potency with GI50 values of <10 nM against 46 out of 53 NCI cell lines [22] but the clinical
trials at phase III stopped due to its high toxicity [23]. Orantinib (21b) (SU6668), which is a more
water soluble indolinone derivative than semaxanib, has significant anti-tumor activity in xenografts
after daily oral treatment [24,25]. SU5614 (21c), in phase II clinical trials, induces growth arrest and
apoptosis in c-kit expressing Kasumi-1, UT-7 and M-07e cells [26].
Incorporation of biphenylurea with an indolin-2-one moiety was proved as a successful strategy
to get active hits to treat hepatocellular carcinoma and to affect cancer cells by inhibition of
tumor-associated carbonic anhydrases [
derivatives 5a and 8a (Figure 1) incorporating indolin-2-one moieties were investigated for their
potential cytotoxic effect towards human breast (MCF-7) and prostate (PC-3) cancer cell lines. Moreover,
4,27]. As an extension of this work, new biphenylurea
–s
,b

Molecules 2016, 21, 762
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physicochemical properties and prediction of ADME descriptors were employed to obtain useful data
about their physicochemical interactions within the body.
2. Results and Discussion
2.1. Chemistry
The synthetic pathway adopted to synthesize the target indolinone derivatives is outlined in
Schemes 1 and 2. Synthesis was initiated by reacting 4-nitrophenyl isocyanate with the appropriate
anilines 1a
in methanol to furnish the corresponding amines 3a
obtained in good yields (75%–81%) through the condensation of the amino intermediates 3a
-
e
to afford 1-(4-nitrophenyl)-3-phenylureas 2a
–e, followed by hydrogenation using Pd/C
–e
. The target indolinone derivatives 5a
–x
were
with
–e
indole-2,3-diones 4a–e in ethyl alcohol in the presence of a catalytic amount of glacial acetic acid
reaction (Scheme 1).
Scheme 1. Synthesis of target compounds 5a–x: Reagents and conditions:
Pd/C, MeOH, rt; iii, EtOH, AcOH (catalytic), reflux 3 h.
i, CH3CN, reflux 2h; ii, H2,
Scheme 2. Synthesis of target compounds 8a
,
b
Reagents and conditions:
i
, DMF, K2CO3, reflux 2h;
ii, EtOH, AcOH (catalytic), reflux 3 h.
Finally, indol-2,3-dione 4a was refluxed with benzyl bromides 6a
,
b
in dry DMF and anhydrous
K2CO3 to yield N-substituted indol-2,3-diones 7a,b, respectively, which were condensed with the key
intermediate 3e in ethyl alcohol in the presence of a catalytic amount of glacial acetic acid to furnish
the target indolinones 8a,b in 77% and 83% yield, respectively (Scheme 2).
The structures of all newly synthesized derivatives were verified on the basis of spectral and
elemental analyses which were in full agreement with the proposed structures.
2.2. Biological Evaluation
The in vitro cytotoxic activity of the prepared compounds 5a–s and 8a,b was examined against
two different cell lines (MCF-7 and PC-3) utilizing the colorimetric assay (SRB) developed by
Skehan et al. [28] using doxorubicin as a reference drug (Table 1).

Molecules 2016, 21, 762
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Table 1. In vitro cytotoxic activities of the synthesized derivatives against MCF-7 and PC-3 cell lines.
IC50 (µM) a
Compd.
R1
R2
R
MCF-7
PC-3
NA b
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
H
H
H
4-F
4-F
H
F
Cl
H
F
Cl
H
F
Cl
H
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
19.53 ˘ 1.05
17.14 ˘ 0.66
19.17 ˘ 1.94
NA b
NA b
20.43 ˘ 1.51
NA b
NA b
NA b
NA b
4-F
48.99 ˘ 3.74
39.53 ˘ 4.02
NA b
NA b
3-CF3
3-CF3
3-CF3
3-Cl
3-Cl
3-Cl
3-Cl
3-Cl
4-Cl
4-Cl
4-Cl
4-Cl
4-Cl
-
51.76 ˘ 4.01
NA b
97.65 ˘ 7.39
NA b
NA b
NA b
F
75.92 ˘ 7.21
1.93 ˘ 0.17
35.59 ˘ 3.27
9.60 ˘ 1.08
1.04 ˘ 0.10
26.02 ˘ 2.51
3.87 ˘ 0.31
12.16 ˘ 1.17
43.08 ˘ 4.22
28.00 ˘ 2.93
4.66 ˘ 0.42
7.30 ˘ 0.84
b
NA b
Cl
Me
OMe
H
73.22 ˘ 5.07
96.00 ˘ 6.85
83.54 ˘ 6.32
NA b
F
NA b
Cl
Me
OMe
-
-
-
NA b
-
-
H
F
-
5s
97.00 ˘ 3.25
18.40 ˘ 1.33
13.79 ˘ 0.88
0.84 ˘ 0.079
8a
8b
Dox.
a
-
-
IC50 values are presented as mean
˘
S.D. of three separate experiments; NA: Compounds having IC50
value > 100 µM.
As shown in Table 1, MCF-7 cells were found to be more susceptible to the synthesized compounds
than prostate cancer (PC-3) cells. The majority of the tested compounds exhibited a competitive
cytotoxic activity against MCF-7 cells. In particular, compounds 5l
, 5o, 5q and 8b exhibited potent
cytotoxic activity against MCF-7, with IC50 = 1.93 0.17, 1.04 0.10, 3.87
or 3.8-, 7-, 1.88- and 1.56-fold increased activity as compared to the reference drug, doxorubicin
˘
˘
˘ 0.31 and 4.66 ˘ 0.42 µM,
(IC50 = 7.3 ˘ 0.84 µM), respectively. Compound 5n possessed cytotoxic activity close to the activity
of the reference drug doxorubicin, with IC50 = 9.60
˘
1.08
µ
M. Compounds 5a
–
c
,
5p
,
5r and 8a were
2.93 M.
also moderately active towards MCF-7 with IC50 values in the range of 12.16
Other analogues showed weak or no activity against MCF-7 cell lines (Table 1).
Concerning activity against PC-3, the synthesized compounds showed moderate to low activity
against this cell line. In particular, compounds 8a were the most active analogues against PC-3 cell
˘
1.17–28.00
˘
µ
,b
line through this study with IC50 values of 18.40 ˘ 1.33 and 13.79 ˘ 0.88 µM, respectively (Table 1).
Regarding the activity against MCF-7 cells, SAR studies provided useful conclusions about
the effects of substitutions on both the indolinone moiety and the terminal phenyl group of the
biphenylurea moiety. Regarding our strategy, we have four different possibilities regarding substitution
depending on whether the substituent is electron donating or electron withdrawing using two different
positions. Herein, electron withdrawing substituents with different types and position were added to
the terminal phenyl group of the biphenylurea moiety, while variable substituents were used on the
indolinone moiety. Concerning substitution on the terminal phenyl group of the biphenylurea moiety,

Molecules 2016, 21, 762
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derivatives with chloro-substituents on either position 3 or 4 produced some derivatives with better
cytotoxic activity than other substituents, as exemplified by compounds 5l 5o 5q and 8b (Table 1). It
was noticed that the substitution on indolinone moiety should be either Cl or no substitution. The other
derivatives such as 5j 5k 5m 5n 5p 5r 5s and 8a, with showed intermediate activity or no activity
,
,
,
,
,
,
,
,
as it may be affected by the 5-substituents on indolinone moiety that are either with no substitution or
with electron donating substitution except compound 5p with 5-F substitution. With no substituents on
the terminal phenyl group of the biphenylurea moiety, compounds 5a–c showed intermediate cytotoxic
activity, while 4-F or 3-CF3 substitution resulted in weak or no activity as in compounds 5d,5i.
Finally, exploration of the impact of the substitution on the N atom of the indolinone suggested
that this was an advantageous approach for activity against PC-3, where the N-substituted members
8a and 8b (IC50 = 18.40
unsubstituted counterpart 5o (IC50 = 83.54
failed to improve the activity against MCF-7 cells (Table 1).
˘
1.33 and 13.79
˘
0.88
µ
M, respectively) showed enhanced activity over their
˘
6.32
µM) towards PC-3. Nevertheless such an approach
2.3. Physicochemical Properties and ADME Profiling
According to Table 2, it is worthy to mention that all of the synthesized compounds (5a–s and 8a,b)
passed the filter of both Lipinski’s rule of five [29] and the Veber rule [30]. The previous fact suggested
all the synthesized compounds possess good oral bioavailability. Additionally, several ADME
descriptors for all the synthesized compounds 5a–s and 8a,b were estimated through a theoretical
kinetic study carried out using the Discovery Studio 2.5 software (Table 3, Accelrys, San Diego,
CA, USA). All of the synthesized compounds showed good predicted levels of human intestinal
absorption except 8a,b that showed moderate intestinal absorption. Regarding aqueous solubility,
all of the tested compounds ranged from very low to low solubility. Concerning blood-brain barrier
penetration, all of the tested compounds were discovered to possess medium penetrability except
compounds 5i, and 8a,b that were undefined. Compounds 5f, 5g, 5h and 5p were predicted to be
CYP2D non-inhibitors.
Table 2. Lipinski rule of five parameters results for all the synthesized compounds.
H-bond
Molecular
Weight
No. of Rotatable
Bonds e
Polar Surface
Area f (Å)
Compd. H-bond Donor a,#
AlogP d
Acceptor b,#
5a
5b
5c
5d
5e
5f
5g
5h
5i
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
3
2
2
6
6
6
6
6
6
6
6
6
6
6
6
6
7
6
6
6
6
7
6
6
356.4
374.4
390.8
374.4
392.4
408.8
424.4
442.4
458.8
390.8
408.8
425.3
404.8
420.8
390.8
408.8
425.3
404.8
420.8
480.9
498.9
3.200
3.405
3.864
3.405
3.611
4.07
4.142
4.348
4.807
3.864
4.07
4.529
4.351
3.848
3.864
4.07
4.529
4.351
3.848
5.654 *
5.859 *
3
3
3
3
3
3
4
4
4
3
3
3
3
4
3
3
3
3
4
5
5
82.59
82.59
82.59
82.59
82.59
82.59
82.59
82.59
82.59
82.59
82.59
82.59
82.59
91.82
82.59
82.59
82.59
82.59
91.82
73.8
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
8a
8b
73.8
a
The upper limit of the number of hydrogen bond donors is 5; b The upper limit of the number of hydrogen
bond acceptors is 10; c The upper limit of the molecular weight is 500; d The upper limit of the AlogP (the log
value of octanol-water partition coefficient) is 500; e The upper limit of the number of rotatable bonds is 10;
* No. of violation allowed is 1 and AlogP value should not be
ě
6; f The upper limit of the polar surface area is
140 Å; # The upper limit of the sum of hydrogen bond donor and acceptors is 12.

Molecules 2016, 21, 762
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Table 3. ADME studies results for all the synthesized compounds.
ADMET
ADMET
ADMET
ADMET BBB
Level d
CYP2D6
Probability
ADMET BBB d
Compd.
CYP2D6 e
Absorption Level a
Solubility b
Solubility Level c
5a
5b
5c
5d
5e
5f
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
´4.438
´4.899
´5.299
´4.878
´5.331
´5.732
´6.048
´6.477
´6.877
´5.289
´5.742
´6.143
´5.734
´5.415
´5.279
´5.732
´6.132
´5.723
´5.404
-6.436
2
2
2
2
2
2
1
1
1
2
2
1
2
2
2
2
1
2
2
1
1
´0.500
´0.436
´0.294
´0.436
´0.373
´0.231
´0.208
´0.145
NA
2
2
2
2
2
2
2
2
4
2
2
2
2
2
2
2
2
2
2
4
4
0.722
0.594
0.584
0.633
0.584
0.485
0.495
0.465
0.544
0.683
0.504
0.514
0.653
0.683
0.623
0.485
0.504
0.643
0.524
0.712
0.762
1
1
1
1
1
0
0
0
1
1
1
1
1
1
1
0
1
1
1
1
1
5g
5h
5i
5j
5k
5l
´0.294
´0.231
´0.089
´0.144
´0.441
´0.294
´0.231
´0.089
´0.144
´0.441
NA
5m
5n
5o
5p
5q
5r
5s
8a
8b
-6.737
NA
a
Human intestinal absorption level. (0 = good, 1 = moderate, 2 = poor, 3 = very poor); b The base 10 logarithm
c
of the molar solubility as predicted by the regression model based by DS; Categorical solubility level.
(0 = extreme low, 1 = very low but possible, 2 = low, 3 = good, 4 = optimal); d Blood brain barrier penetration.
(0;
ě
0.7 = very high penetrant, 1; 0
ď
logBB < 0.7) = High penetrant, 2;
´
0.52 < LogBB < 0 = low, 3;
logBB ď ´0.52 = low, 4; NA = undefiend); e CYP2D inhibition. (0 = non inhibitor, 1 = inhibitor).
3. Experimental Section
3.1. General Information
Melting points were measured with a Stuart melting point apparatus and are uncorrected. Infrared
(IR) spectra were recorded as potassium bromide disks on a FT-IR 8400S spectrophotometer (Shimadzu,
Kyoto, Japan) and values are expressed in wavenumber (cm´1) NMR spectra were recorded on an
1
AV-400 MHz NMR spectrophotometer (Bruker, Karlsruhe, Germany). H-NMR spectra were run at
400 MHz and 13C spectra were run at 100 MHz in deuterated dimethylsulfoxide (DMSO-d6). Chemical
shifts are expressed in
δ values (ppm) using the solvent peak as internal standard. All coupling
constants (J) values are given in Hertz. The abbreviations used to indicate peak multiplicity are as
follows: s, singlet; d, doublet; m, multiplet. Elemental analyses were carried out at the Regional Center
for Microbiology and Biotechnology, Al-Azhar University, Cairo, Egypt. Reaction courses and product
mixtures were routinely monitored by thin layer chromatography (TLC) on silica gel precoated F254
plates Merck (Merck KGaA, Darmstadt, Germany). Unless otherwise noted, all solvents and reagents
were commercially available and used without further purification. Compounds 2a
5o–q [4,31] and 7a,b [32] were all previously prepared.
–e, 3a–e, 5a–l and
3.2. Synthesis
3.2.1. 1-(4-Nitrophenyl)-3-phenyl/substituted phenylurea 2a–e
Compounds 2a–e were prepared according to the literature procedure [3,30].
3.2.2. 1-(4-Aminophenyl)-3-phenyl/substituted phenylurea 3a–e
Compounds 3a–e were prepared according to the literature procedure [3,30].
3.2.3. General Procedure for Preparation of Target Compounds 5a–x
Indoline-2,3-diones 4a
1-(4-aminophenyl)-3-phenyl/substituted phenylurea 3a
–
e
(0.001 mol) were added to a suspension of the appropriate
(0.001 mol) in ethyl alcohol (10 mL)
–
e
containing acatalytic amount of glacial acetic acid. The reaction mixture was heated under reflux for
3 h. The precipitate formed was collected by filtration while hot, washed with hot ethanol, dried and

Molecules 2016, 21, 762
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1
crystallized from ethanol/DMF to afford target compounds 5a–x. H-NMR of 5m, 5n, 5p, 5s, and 8a,b
can be found in the Supplementary Materials.
1-(3-Chlorophenyl)-3-(4-(5-methyl-2-oxoindolin-3-ylideneamino)phenyl)urea (5m): Orange powder (yield 75%)
;
m.p. > 300 ˝C; IR (KBr, cm´1): 3277 (NH), 1739, 1724 (2C=O); 1H-NMR (DMSO-d6)
ν
δ
(ppm): 2.02
and 2.29 (s, 3H, CH3), 6.54 (s, 1H, Ar-H), 6.80 (d, J =7.92 Hz, 1H, Ar-H), 6.80 (d, J = 8.66 Hz, 2H, Ar-H),
7.01–7.43 (m, 4H, Ar-H), 7.56 (d, J = 8.66 Hz, 2H, Ar-H), 7.73 (s, 1H, Ar-H), 8.81 and 8.88 (s, 1H, D2O
exchangeable, NH urea), 8.90 and 8.92 (s, 1H, D2O exchangeable, NH urea), 10.74 and 10.85 (s, 1H,
D2O exchangeable, NH isatin); 13C-NMR (DMSO-d6, 100 MHz)
δ: 21.03, 110.85, 111.75, 112.47, 115.28,
116.29, 117.92, 118.68, 119.79, 123.26, 125.25, 127.25, 129.35, 136.82, 137.33, 143.31, 145.11, 152.70, 154.99,
159.26; Anal. calcd. For C22H17ClN4O2 (404.85): C, 65.27; H, 4.23; N, 13.84; Found C, 65.11; H, 4.27;
N, 13.93.
1-(3-Chlorophenyl)-3-(4-(5-methoxy-2-oxoindolin-3-ylideneamino)phenyl)urea (5n): Red powder (yield 78%);
m.p. > 300 ˝C; IR (KBr,
ν δ (ppm): 3.50
cm´1): 3275 (NH), 1738, 1724 (2C=O); 1H-NMR (DMSO-d6)
and 3.77 (s, 3H, OCH3), 6.19 (s, 1H, Ar-H), 6.80 (d, J = 8.52 Hz, 1H, Ar-H), 6.97–7.44 (m, 6H, Ar-H),
7.56 (d, J = 8.64 Hz, 2H, Ar-H), 7.73 (s, 1H, Ar-H), 8.82 and 8.88 (s, 1H, D2O exchangeable, NH urea),
8.91 and 8.92 (s, 1H, D2O exchangeable, NH urea), 10.66 and 10.77 (s, 1H, D2O exchangeable, NH
isatin); 13C-NMR (DMSO-d6, 100 MHz)
δ: 55.73, 107.60, 111.95, 112.39, 116.70, 118.07, 119.14, 119.88,
121.92, 123.17, 130.86, 133.67, 137.51, 140.78, 141.74, 144.88, 152.91, 154.42, 155.50, 164.20;Anal. calcd.
For C22H17ClN4O3 (420.10): C, 62.79; H, 4.07; N, 13.31; Found C, 62.95; H, 3.99; N, 13.24.
1-(4-Chlorophenyl)-3-(4-(5-methyl-2-oxoindolin-3-ylideneamino)phenyl)urea (5r): Orange powder (yield 81%)
;
m.p. > 300 ˝C; IR (KBr, cm´1): 3273 (NH, NH2), 1739, 1720 (2C=O); 1H-NMR (DMSO-d6)
ν
δ
(ppm):
2.01 and 2.29 (s, 3H, CH3), 6.55 (s, 1H, Ar-H), 6.79 (d, J = 7.90 Hz, 1H, Ar-H), 6.98 (d, J = 8.60 Hz, 2H,
Ar-H), 7.13–7.44 (m, 3H, Ar-H), 7.50 (d, J = 8.78 Hz, 2H, Ar-H), 7.56 (d, J = 8.60 Hz, 2H, Ar-H), 8.78 and
8.87 (s, 1H, D2O exchangeable, NH urea), 8.85 (s, 1H, D2O exchangeable, NH urea), 10.74 and 10.85 (s,
1H, D2O exchangeable, NH isatin); 13C-NMR (DMSO-d6, 100 MHz)
δ: 20.90, 111.73, 116.330, 118.49,
119.35, 119.63, 120.17, 122.19, 128.98, 130.83, 135.11, 137.63, 139.18, 144.71, 145.09, 152.95, 154.92, 164.21;
Anal. calcd. For C22H17ClN4O2 (404.85): C, 65.27; H, 4.23; N, 13.84; Found C, 65.03; H, 4.32; N, 13.93.
1-(4-Chlorophenyl)-3-(4-(5-methoxy-2-oxoindolin-3-ylideneamino)phenyl)urea (5s): Red powder (yield 76%);
m.p. > 300 ˝C; IR (KBr,
ν δ (ppm): 3.49
cm´1): 3283 (NH), 1740, 1724 (2C=O); 1H-NMR (DMSO-d6)
and 3.77 (s, 3H, CH3), 6.20 (s, 1H, Ar-H), 6.81 (d, J = 8.56 Hz, 1H, Ar-H), 6.95–7.16 (m, 3H, Ar-H), 7.32
(d, J = 8.84 Hz, 2H, Ar-H), 7.50 (d, J = 8.84 Hz, 2H, Ar-H), 7.57 (d, J = 8.72 Hz, 2H, Ar-H), 8.86 and 8.94
(s, 1H, D2O exchangeable, NH urea), 8.92 (s, 1H, D2O exchangeable, NH urea), 10.66 and 10.77 (s, 1H,
D2O exchangeable, NH isatin); 13C-NMR (DMSO-d6, 100 MHz)
δ: 55.71, 112.06, 112.38, 115.70, 118.47,
119.16, 120.17, 120.40, 122.28, 125.81, 129.07, 137.71, 139.22, 140.77, 144.73, 152.99, 154.46, 164.23; Anal.
calcd. For C22H17ClN4O3 (420.10): C, 62.79; H, 4.07; N, 13.31; Found C, 62.95; H, 4.11; N, 13.42.
3.2.4. N-Benzylindoline-2,3-diones 7a,b
Compounds 7a,b were prepared according to the literature procedure [31].
3.2.5. General Procedure for Preparation of Target Compounds 8a,b
1-(4-(1-Benzyl-2-oxoindolin-3-ylideneamino)phenyl)-3-(4-chlorophenyl)urea (8a): Orange powder (yield 77%)
;
m.p. > 300 ˝C; IR (KBr, cm´1): 3275 (NH), 1736 (2C=O); 1H-NMR (DMSO-d6)
ν
δ
(ppm): 4.88 and
4.99 (s, 2H, -CH2), 6.77–7.11 (m, 4H, Ar-H), 7.23–7.47 (m, 9H, Ar-H), 7.50 (d, J = 8.84 Hz, 2H, Ar-H),
7.57 (d, J = 8.64 Hz, 2H, Ar-H), 8.83 and 8.87 (s, 1H, D2O exchangeable, NH urea), 8.86 (s, 1H, D2O
exchangeable, NH urea); 13C-NMR (DMSO-d6, 100 MHz)
δ: 43.15, 111.08, 115.94, 118.40, 119.30, 120.20,
122.02, 123.38, 125.32, 127.83, 128.03, 129.11, 129.20, 134.52, 136.40, 137.83, 139.17, 144.64, 147.21, 152.95,
153.94, 163.00; Anal. calcd. For C28H21ClN4O2 (480.14): C, 69.92; H, 4.40; N, 11.65; Found C, 69.75; H,
4.47; N, 11.56.

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1-(4-Chlorophenyl)-3-(4-(1-(4-fluorobenzyl)-2-oxoindolin-3-ylideneamino)phenyl)urea (8b): Yellow powder
(yield 83%); m.p. > 300 ˝C; IR (KBr, cm´1): 3273 (NH), 1738, 1721 (2C=O); 1H-NMR (DMSO-d6)
(ppm): 4.87 and 4.99 (s, 2H, -CH2), 6.77 (d, J = 7.56 Hz, 1H, Ar-H), 6.82 (t, J = 7.52 Hz,1H, Ar-H),
ν
δ
6.98–7.25 (m, 5H, Ar-H), 7.33–7.52 (m, 5H, Ar-H), 7.57 (d, J = 8.60 Hz, 2H, Ar-H), 7.64 (d, J = 8.36 Hz,
2H, Ar-H), 8.83 and 8.87 (s, 1H, D2O exchangeable, NH urea), 8.86 (s, 1H, D2O exchangeable, NH
urea); 13C-NMR (DMSO-d6, 100 MHz)
δ: 42.62, 111.02, 115.87, 116.08, 119.31, 119.66, 120.23, 122.92,
125.33, 125.86, 129.10, 129.97, 133.89, 137.84, 139.16, 144.62, 147.05, 152.95, 153.90, 157.60, 160.82, 163.00;
Anal. calcd. For C28H20ClFN4O2 (498.94): C, 67.40; H, 4.04; N, 11.23; Found C, 67.59; H, 4.11; N, 11.32.
3.3. In Vitro Cytotoxic Activity
3.3.1. Cell Culture
Human breast cancer (MCF-7) and prostate cancer (PC-3) cells were purchased from National
Cancer Institute (Cairo, Egypt). Maintenance of cells was carried out using Roswell Park
Memorial Institute medium (RPMI-1640) supplemented with 10% heat-inactivated fetal bovine serum,
100 Units/mL of penicillin and 100 µg/mL streptomycin. Cells were kept as a monolayer culture by
˝
serial passaging under standard culture conditions of 37 C, 95% humidified air and 5% CO2.
3.3.2. In vitro Cytotoxic Activity
Investigated compounds were dissolved in DMSO and kept at a stock concentration of
100 mM. Cytotoxicity was assessed via SRB method as indicated by Skehan et al. [28] In brief,
exponentially growing cells were collected using 0.25% Trypsin-EDTA and seeded in 96-well plates at a
3000 cells/well density in culture medium and incubated for 24 h till the formation of a monolayer.
Subsequently, cells were incubated with different concentrations (0–103
µM) of the compounds under
investigation as well as doxorubicin as a reference drug for further 72 h. At the end of the incubation,
˝
the treatment was terminated through fixing the cells with 10% trichloroacetic acid for 1 h at 4 C. This
was followed by a staining procedure using 0.4% SRB (1% acetic acid) for 10 min at room temperature.
Finally, microplates were left to dry for 24 h and Tris-HCl was added afterwards for 5 min on a shaker
at 1600 rpm for solubilization. Using an ELISA microplate reader (ChroMate-4300, Palm City, FL,
USA), optical density was determined at 545 nm. The IC50 values were calculated according to the
equation for Boltzman sigmoidal concentration–response curve using the nonlinear regression fitting
models (Graph Pad Prism Version 5; GraphPad Software, Inc. La Jolla, CA, USA). The results reported
are means of at least three separate experiments. Statistical analyses were carried out using one-way
analysis of variance (ANOVA) followed by the Tukey-Kramer test for post-hoc analysis. Statistical
difference was considered to be significant at p < 0.05. Statistical testing was performed using GraphPad
InStat software, version 3.05.
3.3.3. Physicochemical Properties and ADME Profiling
Physicochemical properties and ADME profiling for all the synthesized compounds were
performed using Discovery Studio 4 (Accelrys, San Diego, CA, USA). All the tested compounds were
drawn as a small library and prepared using prepare ligand protocol to find the suitable orientation
in 3D. For studying physicochemical properties, the prepared library was filtered using the Lipinski
and Veber rules protocols. ADME profiling was predicted for the designed library using ADME
descriptors protocol.
4. Conclusions
We reported herein the synthesis, in vitro cytotoxic activity and in silico ADME prediction studies
of twenty one biphenylurea derivatives 5a
synthesized compounds were evaluated for their in vitro cytotoxicity against two human cancer cell
lines, namely MCF-7 breast cancer and PC-3 prostate cancer. Compounds 5l 5o 5q and 8b exhibited
–s and 8a,b that incorporated an indolinone moiety. All the
,
,

Molecules 2016, 21, 762
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potent cytotoxic activity against MCF-7 with IC50 = 1.93, 1.04, 3.87 and 4.66
µM, respectively and all of
these compounds were found to be more active than the reference drug doxorubicin (IC50 = 7.3 µM)
.
All of the tested compounds were filtered according to the Lipinski and Veber rules and all of them
passed the filter. In addition they showed varied physicochemical properties according to their
ADME descriptors.
Supplementary Materials: Supplementary materials can be accessed at: http://www.mdpi.com/1420-3049/21/
6/762/s1.
Acknowledgments: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University,
Cairo, Egypt, is highly appreciated for supporting this research. The authors would like to extend their sincere
appreciation to the Deanship of Scientific Research at King Saud University for its funding of this research through
the Research Group Project no. PRG-1436-038.
Author Contributions: Wagdy M. Eldehna and Hatem A. Abdel-Aziz conceived and designed the experiments;
Wagdy M. Eldehna, Mohamed Fares, Hany S. Ibrahim and Muhammad A. Alsherbiny carried out the experiments;
Muhammad A. Alsherbiny, and Hazem A. Ghabbour analyzed and interpreted the data; Mohamed Fares,
Hany S. Ibrahim prepared the manuscript; Mohamed H. Aly performed the biological screening. All authors have
read and approved the final manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
2.
3.
World Cancer Report 2014. Available online: http://www.iarc.fr/en/publications/books/wcr/wcr-order.
php (accessed on 6 May 2016).
Torre, L.A.; Bray, F.; Siegel, R.L.; Ferlay, J.; Lortet-Tieulent, J.; Jemal, A. Global cancer statistics, 2012.
CA-Cancer J. Clin. 2015, 65, 87–108. [CrossRef] [PubMed]
Havrylyuk, D.; Kovach, N.; Zimenkovsky, B.; Vasylenko, O.; Lesyk, R. Synthesis and Anticancer Activity
of Isatin-Based Pyrazolines and Thiazolidines Conjugates. Arch. Pharm. 2011, 344, 514–522. [CrossRef]
[PubMed]
4.
Eldehna, W.M.; Fares, M.; Ibrahim, H.S.; Aly, M.H.; Zada, S.; Ali, M.M.; Abou-Seri, S.M.; Abdel-Aziz, H.A.;
El Ella, D.A.A. Indoline ureas as potential anti-hepatocellular carcinoma agents targeting VEGFR-2: Synthesis,
in vitro biological evaluation and molecular docking. Eur. J. Med. Chem. 2015, 100, 89–97. [CrossRef]
[PubMed]
5.
6.
7.
8.
9.
Eldehna, W.M.; Ibrahim, H.S.; Abdel-Aziz, H.A.; Farrag, N.N.; Youssef, M.M. Design, synthesis and in vitro
antitumor activity of novel N-substituted-4-phenyl/benzylphthalazin-1-ones. Eur. J. Med. Chem. 2015, 89,
549–560. [CrossRef] [PubMed]
Ibrahim, H.S.; Abou-seri, S.M.; Ismail, N.S.; Elaasser, M.M.; Aly, M.H.; Abdel-Aziz, H.A. Bis-isatin
hydrazones with novel linkers: Synthesis and biological evaluation as cytotoxic agents. Eur. J. Med.
Chem. 2016, 108, 415–422. [CrossRef] [PubMed]
˙
Sławin´ski, J.; Grzonek, A.; Zołnowska, B.; Kawiak, A. Synthesis of Novel Pyrido[4,3-e][1,2,4]
triazino[3,2-c][1,2,4]thiadiazine 6,6-dioxide Derivatives with Potential Anticancer Activity. Molecules 2015
21, 41. [CrossRef] [PubMed]
,
Fares, M.; Eldehna, W.M.; Abou-Seri, S.M.; Abdel-Aziz, H.A.; Aly, M.H.; Tolba, M.F. Design, Synthesis and
in Vitro Antiproliferative Activity of Novel Isatin-Quinazoline Hybrids. Arch. Pharm. 2015, 348, 144–154.
[CrossRef] [PubMed]
Wilhelm, S.; Carter, C.; Lynch, M.; Lowinger, T.; Dumas, J.; Smith, R.A.; Schwartz, B.; Simantov, R.; Kelley, S.
Discovery and development of sorafenib: a multikinase inhibitor for treating cancer. Nat. Rev. Drug Discov.
2006, 5, 835–844. [CrossRef] [PubMed]
10. Woo, H.Y.; Heo, J. Sorafenib in liver cancer. Expert. Opin. Pharmacother. 2012, 13, 1059–1067. [CrossRef]
[PubMed]
11. Wilhelm, S.; Dumas, J.; Ladouceur, G.; Lynch, M.; Scott, W. Diaryl Ureas with Kinase Inhibiting Activity. U.S.
Patent 20070020704, 25 January 2007.
12. Wilhelm, S.M.; Dumas, J.; Adnane, L.; Lynch, M.; Carter, C.A.; Schutz, G.; Thierauch, K.H.; Zopf, D.
Regorafenib (BAY 73–4506): A new oral multikinase inhibitor of angiogenic, stromal and oncogenic receptor

Molecules 2016, 21, 762
10 of 11
tyrosine kinases with potent preclinical antitumor activity. Int. J. Cancer 2011, 129, 245–255. [CrossRef]
[PubMed]
13. DiGiulio, S. FDA Approves Stivarga for Advanced GIST. Oncol. Times 2013, 35, 12.
14. Tan, E.-H.; Goss, G.D.; Salgia, R.; Besse, B.; Gandara, D.R.; Hanna, N.H.; Yang, J.C.-H.; Thertulien, R.;
Wertheim, M.; Mazieres, J. Phase 2 trial of Linifanib (ABT-869) in patients with advanced non-small cell lung
cancer. J. Thorac. Oncol. 2011, 6, 1418–1425. [CrossRef] [PubMed]
15. Supuran, C.T.; Winum, J.-Y. Designing carbonic anhydrase inhibitors for the treatment of breast cancer.
Expert Opin. Drug Discov. 2015, 10, 591–597. [CrossRef] [PubMed]
16. Lomelino, C.L.; Mahon, B.P.; McKenna, R.; Carta, F.; Supuran, C.T. Kinetic and X-ray crystallographic
investigations on carbonic anhydrase isoforms I, II, IX and XII of a thioureido analog of SLC-0111.
Bioorg. Med. Chem. 2016, 24, 976–981. [CrossRef] [PubMed]
17. Vine, K.; Matesic, L.; Locke, J.; Ranson, M.; Skropeta, D. Cytotoxic and anticancer activities of isatin and
its derivatives: a comprehensive review from 2000–2008. Anti-Cancer Agents Med. Chem. 2009, 9, 397–414.
[CrossRef]
18. Vine, K.L.; Matesic, L.; Locke, J.M.; Skropeta, D. Recent highlights in the development of isatin-based
anticancer agents. Adv. Anticancer Agents Med. Chem. 2013, 2, 254–312.
19. Motzer, R.J.; Michaelson, M.D.; Redman, B.G.; Hudes, G.R.; Wilding, G.; Figlin, R.A.; Ginsberg, M.S.;
Kim, S.T.; Baum, C.M.; DePrimo, S.E. Activity of SU11248, a multitargeted inhibitor of vascular endothelial
growth factor receptor and platelet-derived growth factor receptor, in patients with metastatic renal cell
carcinoma. J. Clin. Oncol. 2006, 24, 16–24. [CrossRef] [PubMed]
20. Prenen, H.; Cools, J.; Mentens, N.; Folens, C.; Sciot, R.; Schöffski, P.; Van Oosterom, A.; Marynen, P.;
Debiec-Rychter, M. Efficacy of the kinase inhibitor SU11248 against gastrointestinal stromal tumor mutants
refractory to imatinib mesylate. Clin. Cancer Res. 2006, 12, 2622–2627. [CrossRef] [PubMed]
21. London, C.A.; Malpas, P.B.; Wood-Follis, S.L.; Boucher, J.F.; Rusk, A.W.; Rosenberg, M.P.; Henry, C.J.;
Mitchener, K.L.; Klein, M.K.; Hintermeister, J.G. Multi-center, placebo-controlled, double-blind, randomized
study of oral toceranib phosphate (SU11654), a receptor tyrosine kinase inhibitor, for the treatment of dogs
with recurrent (either local or distant) mast cell tumor following surgical excision. Clin. Cancer Res. 2009, 15,
3856–3865. [CrossRef] [PubMed]
22. Pandit, B.; Sun, Y.; Chen, P.; Sackett, D.L.; Hu, Z.; Rich, W.; Li, C.; Lewis, A.; Schaefer, K.; Li, P.-K.
Structure–activity-relationship studies of conformationally restricted analogs of combretastatin A-4 derived
from SU5416. Bioorg. Med. Chem. 2006, 14, 6492–6501. [CrossRef] [PubMed]
23. Griffith, R.; Brown, M.; McCluskey, A.; Ashman, L. Small molecule inhibitors of protein kinases in cancer-how
to overcome resistance. Mini Rev. Med. Chem. 2006, 6, 1101–1110. [CrossRef] [PubMed]
24. Sessa, C.; Viganò, L.; Grasselli, G.; Trigo, J.; Marimon, I.; Lladò, A.; Locatelli, A.; Ielmini, N.; Marsoni, S.;
Gianni, L. Phase I clinical and pharmacological evaluation of the multi-tyrosine kinase inhibitor SU006668
by chronic oral dosing. Eur. J. Cancer 2006, 42, 171–178. [CrossRef] [PubMed]
25. Laird, A.D.; Vajkoczy, P.; Shawver, L.K.; Thurnher, A.; Liang, C.; Mohammadi, M.; Schlessinger, J.; Ullrich, A.;
Hubbard, S.R.; Blake, R.A. SU6668 is a potent antiangiogenic and antitumor agent that induces regression of
established tumors. Cancer Res. 2000, 60, 4152–4160. [PubMed]
26. Spiekermann, K.; Faber, F.; Voswinckel, R.; Hiddemann, W. The protein tyrosine kinase inhibitor SU5614
inhibits VEGF-induced endothelial cell sprouting and induces growth arrest and apoptosis by inhibition of
c-kit in AML cells. Exp. Hematol. 2002, 30, 767–773. [CrossRef]
27. Eldehna, W.M.; Fares, M.; Ceruso, M.; Ghabbour, H.A.; Abou-Seri, S.M.; Abdel-Aziz, H.A.;
El Ella, D.A.A.; Supuran, C.T. Amido/ureidosubstituted benzenesulfonamides-isatin conjugates as low
nanomolar/subnanomolar inhibitors of the tumor-associated carbonic anhydrase isoform XII. Eur. J. Med.
Chem. 2016, 110, 259–266. [CrossRef] [PubMed]
28. Skehan, P.; Storeng, R.; Scudiero, D.; Monks, A.; McMahon, J.; Vistica, D.; Warren, J.T.; Bokesch, H.; Kenney, S.;
Boyd, M.R. New colorimetric cytotoxicity assay for anticancer-drug screening. J. Natl. Cancer. Inst. 1990, 82,
1107–1112. [CrossRef] [PubMed]
29. Lipinski, C.A.; Lombardo, F.; Dominy, B.W.; Feeney, P.J. Experimental and computational approaches to
estimate solubility and permeability in drug discovery and development settings. Adv. Drug Deliv. Rev. 2012
,
64, 4–17. [CrossRef]

Molecules 2016, 21, 762
11 of 11
30. Veber, D.F.; Johnson, S.R.; Cheng, H.-Y.; Smith, B.R.; Ward, K.W.; Kopple, K.D. Molecular properties that
influence the oral bioavailability of drug candidates. J. Med. Chem. 2002, 45, 2615–2623. [CrossRef] [PubMed]
31. Eldehna, W.M.; Abou-Seri, S.M.; El Kerdawy, A.M.; Ayyad, R.R.; Hamdy, A.M.; Ghabbour, H.A.; Ali, M.M.;
El Ella, D.A.A. Increasing the binding affinity of VEGFR-2 inhibitors by extending their hydrophobic interaction
with the active site: Design, synthesis and biological evaluation of 1-substituted-4-(4-methoxybenzyl)
phthalazine derivatives. Eur. J. Med. Chem. 2016, 113, 50–62. [CrossRef] [PubMed]
32. Presset, M.; Mohanan, K.; Hamann, M.; Coquerel, Y.; Rodriguez, J. 1, 3-Dipolar cycloaddition of hydrazones
with α-oxo-ketenes: A three-component stereoselective entry to pyrazolidinones and an original class of
spirooxindoles. Org. Lett. 2011, 13, 4124–4127. [CrossRef] [PubMed]
Sample Availability: Samples of all compounds are available from the authors.
©
2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC-BY) license (http://creativecommons.org/licenses/by/4.0/).